The Central Nervous System (CNS) has long been considered to be a site of relative immune privilege. However, it is increasingly recognized that CNS tissue injury in acute and chronic neurological disease may be mediated by the CNS inflammatory response. The CNS inflammatory response is primarily mediated by inflammatory cytokines.
The microglial cell is the primary immunocompetent cell in the central nervous system. Microglia are morphologically, immunophenotypically and functionally related to cells of the monocyte/macrophage lineage (Gehrmenn et al., 1995). Acute CNS insult, as well as chronic conditions such as HIV encephalopathy, epilepsy, and Alzheimer's disease (AD) are associated with microglial activation (McGeer et al., 1993; Rothwell and Relton, 1993; Giulian et al., 1996; Sheng et al., 1994). Microglial activation results in the production of nitric oxide (NO) and other free radical species, and the release of proteases, inflammatory cytokines (including IL-1β, IL-6 and TNFα), and a neurotoxin that works through the NMDA receptor (Giulian et al., 1996). Microglial activation can be assessed by measuring the production of nitrite, a stable product of nitric oxide formation (Barger and Harmon, 1997).
Apolipoprotein E (ApoE) plays a role in cholesterol metabolism and has also been reported to have immunomodulatory properties. For instance, ApoE has been demonstrated to have immunomodulatory effects in vitro, including suppression of lymphocyte proliferation and immunoglobulin synthesis after mitogenic challenge (Avila et al., 1982; Edgington and Curtiss, 1981). ApoE is secreted in large quantities by macrophage after peripheral nerve injury, and by astrocytes and oligodendrocytes (glial cells) after CNS injury (Stoll et al., 1989; Stoll and Mueller, 1986). The role that ApoE plays in glial activation and CNS injury, however, remains controversial.
The majority of ApoE is produced in the liver. However, due to its large size, ApoE does not readily cross the blood-brain barrier. In fact, following liver transplantation, peripheral apoE phenotype changes to that of the donor liver, while CSF (cerebrospinal fluid) apoE phenotype remains unchanged (Linton et al., 1991). Thus, ApoE localized within the nervous system represents a discrete pool from protein produced in the periphery (Laskowitz et al., January 2001).
ApoE is a 299 amino acid lipid-carrying protein with a known sequence (Rail et al., J. Biol. Chem. 257:4174 (1982); McLean et al., J. Biol. Chem. 259:6498 (1984)). The complete gene for human ApoE has also been sequenced (Paik et al., Proc. Natl. Acad. Sci. USA 82:3445 (1985). ApoE sequences from at least ten species have been determined, and show a high degree of conservations across species, except at the amino and carboxyl termini. Weisgraber, Advances in Protein Chemistry 45:249 (1994).
Human ApoE is found in three major isoforms: ApoE2, ApoE3, and ApoE4; these isoforms differ by amino acid substitutions at positions 112 and 158. The most common isoform is ApoE3, which contains cysteine at residue 112 and arginine at residue 158; ApoE2 is the least common isoform and contains cysteine at residues 112 and 158; ApoE4 contains arginine at residues 112 and 158. Additional rare sequence mutations of human ApoE are known (see, e.g., Weisgraber, Advances in Protein Chemistry 45:249 (1994), at page 268-269).
ApoE has two distinct functional domains, a 10-kDa carboxyl terminus and a 22 k-Da amino terminus (Wetterau et al., 1988). The carboxyl terminus has a high affinity for lipid and mediates the role of ApoE in cholesterol transport. The amino terminus is composed of four antiparallel alpha helices, which includes the receptor binding region (Weisbarger et al., 1983; Innerarity et al., 1983). ApoE is known to bind a family of cell surface receptors, including the LDL, VLDL, LRP/α2M, ER-2, LR8 receptors, apoE receptor 2 (apoER2), and megalin/gp330 (Kim et al., 1996; Novak et al., 1996; Veinbergs et al., 2001). The interaction of apolipoprotein E and the LDL receptor is important in lipoprotein metabolism. In studies of the LDL receptor-binding activity of ApoE, it is typically complexed with phospholipid. The protein has been described as essentially inactive in the lipid-free state (Innerarity et al., 1979).
One region of ApoE which is critical for the interaction with the LDL receptor lies between residues 140-160 (Mahley, 1988), and site-specific mutagenesis studies of this region have demonstrated that mutations affecting charge and conformation can result in defective binding (Lalazar, 1988). The receptor binding region of ApoE (i.e., amino acid residues 135-160) is rich in basic amino acids including arginine and lysine. Various amino acid substitutions in the receptor binding region of ApoE have been studied for their effects on ApoE-LDL receptor binding. Substitution of either arginine or lysine at residues 136, 142, 145 and 146 with neutral residues decreased normal ApoE3 binding activity (Weisgraber, 1988). No single substitution of a basic residue within the receptor-binding region of ApoE3 completely disrupts LDL receptor binding, suggesting that no one residue is critical for this interaction. It has been postulated that regions of ApoE outside the LDL binding region are necessary to maintain the receptor-binding region in an active binding conformation (Weisgraber, 1994). Dyer et al. (1991) studied lipid-free synthetic peptide fragments comprising residues 141-155 of ApoE, and a dimeric peptide of this sequence. No binding activity was observed with the monomer of this peptide, but low levels of binding were observed with the dimer (˜1% of LDL activity).
The receptors that bind ApoE have areas of high sequence similarity. The scavenger receptor is known to be present on microglia, and preferentially binds acetylated and oxidized LDL. The scavenger receptor may be particularly relevant under inflammatory (oxidizing) conditions. Scavenger receptors are also known to be upregulated in microglia after injury (Bell et al., 1994).
LRP receptors are known to be present on macrophages. In overview, following modification by lipoprotein lipase and the association of apolipoproteins, very large density lipoproteins (VLDL) and chylomicron become remnants, and are cleared hepatically by a receptor-mediated mechanism. Although recognized as distinct from the low density lipoprotein (LDL) receptor, the remnant receptor also has a high affinity for ApoE, and recognizes the remnant particles via incorporated ApoE moieties. In 1988, this remnant receptor was cloned, and dubbed the LDL receptor-related related protein, or “LRP”.
The LRP is a large receptor, with a primary sequence of 4525 amino acids, and bears many structural similarities to other members of the LDL receptor family. Like the LDL receptor, the extracellular domain of LRP includes a cysteine-enriched ligand binding domain and EGF precursor homology domain which are believed to play a role in the acid-dependent dissociation of ligand from the receptor. Unlike the LDL receptor, however, the O-lined sugar domain is not present in the extracellular portion adjacent to the membrane. As with all of the members of the LDL receptor family, LRP is a transmembrane protein, and is anchored by a single transmembrane segment. The cytoplasmic tail of the protein is 100 amino acids, approximately twice as long as the LDL receptor, and contains the NpxY motif, which is believed to be necessary for targeted coated-pit mediated endocytosis (Krieger and Herz, 1994; Misra et al., 1994).
In addition to binding lipid, ApoE also binds lipopolysaccharide (LPS), which is an endotoxin that mediates Gram-negative sepsis by inducing the production of macrophage-derived cytokines. These cytokines, which include TNFα, IL-1α, IL-1β and IL-6, are responsible for the metabolic and physiologic changes that ultimately lead to pathology (Waage et al., 1987; Chensue et al., 1991: Henderson et al., 1996). ApoE redirects bound LPS from macrophages to parenchymal liver cells, which mediate the subsequent secretion of LPS into the bile where it is inactivated (Harris et al., 1993; Harris et al., 1998). Consequently, macrophages become less activated and produce less of the proinflammatory mediators.
Laskowitz et al. (June 1997) described experiments in which mixed neuronal-glial cell cultures from apoE-deficient mice were stimulated with lipopolysaccharide (LPS). It was found that preincubation of the cell cultures with apoE blocked glial secretion of TNFα in a dose-dependent manner. More recently, Van Oosten et al. demonstrated that concomitant administration of ApoE with a lethal dose of LPS protected mice against LPS-induced mortality (Van Oosten et al., 2001). Rensen et al. demonstrated that a free ApoE molecule binds approximately two molecules of LPS, possibly by an exposed hydrophilic domain involving arginine residues since selective elimination of the positive charge on arginine residues of apoE resulted in a largely reduced binding of LPS to ApoE and abolished the effect of ApoE on the in vivo behavior of LPS (Rensen et al., 1997). Interestingly, lactoferrin is a glycoprotein with an arginine/lysine-rich sequence at positions 25-31 resembling the receptor binding site (amino acids 142-148) of ApoE, and has also been shown to bind LPS (Huettinger et al., 1988; Cohen et al., 1992; Miyazawa et al., 1991). Although, he showing by Laskowitz et al. that preincubation of ApoE with neuronal-glial cell cultures blocked LPS-induced TNF-alpha tion whereas coadministration of ApoE with LPS did not suggests that some other mechanism than LPS binding is involved (Laskowitz et al., June 1997).
In addition to its roles in cholesterol metabolism and endotoxin clearance, ApoE may also play an important role in neurological disease. The presence of ApoE4 has been associated with risk of developing sporadic and late-onset Alzheimer's disease (Strittmatter et al., 1993). Barger and Harmon (August 1997) reported that treatment of microglia with a secreted derivative of beta-amyloid precursor protein (sAPP-alpha) activated microglia, induced inflammatory reactions in microglia, and enhanced the production of neurotoxins by microglia. The ability of sAPP-alpha to activate microglia was blocked by prior incubation of the sAPP-alpha protein with apolipoprotein E3 but not apolipoprotein E4. More recently, some researchers have proposed an involvement of ApoE in regulating Tau phosphorylation, suggesting that ApoE is involved some way in the development of the neurofibrillary fibrils associated with Alzheimer's Disease (Flaherty et al., 1999; Tesseur et al., 2000). However, the link between ApoE and Tau has remained controversial (Lovestone, 2001).
There have also been numerous clinical and experimental observations demonstrating that ApoE modifies the response of brain to acute injury. For example, clinical observations suggest that the ApoE4 allele is associated with increased mortality and functional deficit after acute and chronic closed head injury (Sorbi et al., 1995; Teasdale et al., 1997; Jordan et al., 1997; Friedman et al., 1999). The ApoE4 allele has also been associated with the extent of amyloid β-protein deposition following head injury (Mayeux et al., 1995; Nicoll et al., 1995).
The deleterious effects of the apoE4 isoform on neurological outcomes have also been observed in a variety of clinical settings associated with cerebral ischemia. These include stroke (Slooter et al., 1997), intracranial hemorrhage (Alberts et al., 1995; McCarron et al., 1998), cognitive deficit after cardiopulmonary bypass (Tardiff et al., 1997) and hypoxic brain injury following cardiac arrest resuscitation (Schiefermeier et al., 2000). The role of ApoE following focal ischemia is less clear, however, with at least one clinical study failing to document an effect of apoE genotype on functional outcome following stroke (Broderick et al., 2001).
Clinical observations implicating a role for apoE in modifying the central nervous system response to ischemia have recently been extended to animal models. ApoE deficient mice have larger infarcts and worse functional outcomes following focal ischemia and reperfusion relative to control animals matched for age, sex, and genetic background (Laskowitz et al., July 1997).
This effect is independent of cerebral blood flow or cerebrovascular anatomy (Bart et al., 1998). In models of transient forebrain ischemia, apoE deficient animals also have increased injury to neuronal populations that are selectively vulnerable to cerebral hypoperfusion, including hippocampus, caudoputamen, and cortex (Sheng et al., 1999; Horsburgh et al., 1999). This increased sensitivity to ischemia can be reversed by intraventricular administration of human recombinant apoE (Horsburgh et al., 2000). Moreover, consistent with the clinical literature, apoE deficient mice expressing the human apoE4 transgene have larger infarcts and worse functional outcomes than mice expressing the human apoE3 transgene (Sheng et al., 1998).
Although there are multiple clinical reports demonstrating that apoE genotype influences neurological recovery in isoform-specific fashion, the mechanisms by which this occur remain poorly defined. It has been proposed that endogenous apoE may influence the CNS response to injury by modifying oxidative stress (Miyata and Smith, 1996), exerting direct neurotrophic effects (Holtzman et al., 1995), downregulating the CNS inflammatory response (Lynch et al., 2001), or serving as a pathological chaperone by promoting cerebral amyloid deposition (Wisniewski and Frangione, 1992). More recent studies, however, have failed to demonstrate any neuroprotective effect from the intact ApoE protein (Jordan et al., 1998; Lendon et al., 2000).
Furthermore, several studies have suggested that ApoE derived peptide fragments may cause neuronal injury. For example, it has recently been demonstrated that carboxyl-terminal truncated forms of apoE occur in the brains of patients with AD, presumably as a result of intracellular processing. These fragments are bioactive and are capable of interacting with cytoskeletal proteins to induce inclusions resembling neurofibrillary tangles in cultured neurons (Huang et al., 2001). Moulder et al. recently reported that a dimer composed of the ApoE-derived peptide 141-155 has a neurotoxic effect, suggesting to the authors that ApoE itself could be a source of toxicity in Alzheimer's disease brain (Moulder et al., 1999). Using a peptide comprised of a tandem repeat of residues 141-149, Tolar et al. demonstrated that exposure of primary hippocampal neurons to this peptide induced neuronal cell death, an effect which was blocked by preincubation with MK-801, an NMDA antagonist (Tolar et al., 1999). These results predict that exposure with this tandem repeat peptide amplifies NMDA-induced excitotoxicity by direct or indirect mechanisms.
In summary, ApoE plays varied roles in different biological processes. While ApoE appears to provide a protective effect in the periphery by removing LPS from macrophages, the role it plays in CNS injury and neurological diseases such as Alzheimer's Disease is far from clear. What is needed is a better understanding of how ApoE contributes to the CNS inflammatory response, to aide in the formulation of reagents for use in the treatment of neurological injury and disease.